Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 285 (6), 4015-24

Calmodulin Wraps Around Its Binding Domain in the Plasma Membrane Ca2+ Pump Anchored by a Novel 18-1 Motif

Affiliations

Calmodulin Wraps Around Its Binding Domain in the Plasma Membrane Ca2+ Pump Anchored by a Novel 18-1 Motif

Nenad Juranic et al. J Biol Chem.

Abstract

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

Figures

FIGURE 1.
FIGURE 1.
Schematic representation of the PMCA and peptide sequences from the CBD of its C-tail. The model on top shows the PMCA in the autoinhibited form where the CBD interacts with both the first and second intracellular loop. The C-tail of 150 amino acids (a.a) beginning after the last membrane-spanning domain is indicated in red. The sequences and nomenclature of peptides used to study the CaM-CBD interaction are shown on the bottom.
FIGURE 2.
FIGURE 2.
A, Superposition of (1H-15N) heteronuclear single quantum coherence spectra of holoCaM in complex with C28 (black) and free holoCaM (red). Pronounced shifts exhibited by residues Thr-29, Val-55, Ala-57, Met-71, Phe-92, Met-144, and Ala-147 are indicated by arrows. B, comparison of the CaM chemical shift difference (ΣΔ = |δ1Hcomplex − δ1Hfree| + |δ15Ncomplex − δ15Nfree|) for the C28, C20, and M13 complexes. The peak positions of holoCaM in complex with C20 are taken from Ref. , and those for the complex with M13 peptide were taken from Ref. .
FIGURE 3.
FIGURE 3.
Distribution of the normalized 1DHN, 1DC′Cα, and 1DC′N RDCs from holoCaM·C28 (black) compared with the RDC distribution from the C-terminal domain alone (gray).
FIGURE 4.
FIGURE 4.
Selected NMR data on the 13C,15N-labeled C28 peptide in the holoCaM·C28 complex obtained on a 700-MHz Bruker Avance spectrometer at 298 K. A, RDC “wave” of sequential 1DNH from the C28 peptide; comparison of experimental values (solid line) with those calculated for an ideal helix (dashed line). B, sequential peptide backbone {1H,15N} NOE. C, amide region two-dimensional {1H,15N} TROSY spectrum with the residue numbers according to their position in PMCA4b (the Trp-1093-Hϵ1 signal is from the Trp-1093 side chain indole).
FIGURE 5.
FIGURE 5.
Backbone Cα traces (stereo view) of the ensemble of 20 conformers representing the holoCaM·C28 structure (top panel), with two views of the ribbon model of the most representative conformer from the same ensemble (middle and bottom panels). Side chains of the peptide anchoring residues Trp-1093 (Trp-8) and Phe-1110 (Phe-25) are also shown. The CaM N-terminal domain is colored blue, the C-terminal domain is red, and C28 peptide is green.
FIGURE 6.
FIGURE 6.
Positioning of CaM lobes around the helical peptide in the anti-parallel Phe-Trp type anchoring. The dihedral angle Ω is defined by the Cα atoms of residues Ile-27(CaM)-Phe-Trp-Val-136(CaM), and distance L is between the Cα atoms of the Trp and Phe anchors. The diagram on the bottom right shows the dependence of Ω and L on the relative position of the second anchor (Δn+1), using data of structures (21, 24, 31, 32, 36–45) in supplemental Table S1 (solid circles denote cases of the anti-parallel Phe-Trp type anchoring). The values expected for an ideal αD-helix are represented by the solid line. The “forbidden” positions are marked by ×.
SCHEME 1.
SCHEME 1.
Sequence comparison of peptides bound by CaM via the Phe-Trp anchoring mode. The anchor positions are indicated (Anch.). Conserved hydrophobic and hydrophilic residue positions are boxed.
FIGURE 7.
FIGURE 7.
Space filling models of the holoCaM lobes (N-terminal lobe in blue, C-terminal lobe in red, peptide in green) from the four structures representing the four types of lobe anchoring to a helical peptide. The left column structures (without peptide ligand) show the lobe contacts; the right column structures (with ligand) show peptide exposure.
FIGURE 8.
FIGURE 8.
Overlay (stereo view) of representative structure from the holoCaM·C28 ensemble (model 1 in our Protein Data Bank deposition 2kne) and representative (top) or rare (bottom) structures from the holoCaM·C20 ensemble (models 1 and 5 from Protein Data Bank entry 1cff (7)). Structures are aligned in their C-terminal domain (red). The N-terminal domains of holoCaM in the C28 and C20 complex are color-coded dark and light blue, respectively. The peptides C28 and C20 are color-coded green and magenta, respectively.

Similar articles

See all similar articles

Cited by 13 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Associated data

Feedback